CN108251486A

CN108251486A - The preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang

Info

Publication number: CN108251486A
Application number: CN201810257264.3A
Authority: CN
Inventors: 姜建辉; 赵俭波; 丁慧萍; 辛文博; 高来福
Original assignee: Tarim University
Current assignee: Tarim University
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2018-07-06
Anticipated expiration: 2038-03-27
Also published as: CN108251486B

Abstract

The present invention discloses the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang, includes the following steps：(1) the black bee royal jelly freeze-dried powder in Xinjiang is prepared；(2) degreasing and the main albumen of royal jelly is prepared；(3) globulin is prepared；(4) the black bee royal jelly globulin enzymolysis product in Xinjiang is prepared；(5) different molecular weight peptide fragment in globulin enzymolysis product is detached.By extracting globulin in the black bee royal jelly in Xinjiang, then it is digested, different molecular weight peptide fragment is classified as after obtaining enzymolysis product, using the antioxidant activity removed the assay method of the bitter hydrazine free radical (DPPH) of diphenyl 2, Linoleic Acid Oxidation method is inhibited to survey royal jelly globulin and its enzymolysis product, and its total antioxidant capacity is surveyed, obtain the most strong peptide matters less than 3kD of antioxidant activity.

Description

The preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang

Technical field

The present invention relates to the researchs of the black bee royal jelly globulin in Xinjiang.More particularly, to the black bee in antioxidant activity Xinjiang The preparation method of royal jelly globulin enzymolysis product.

Background technology

The black bee in Xinjiang also known as the black bees of Yi Li belong to a strain with Apis mellifera mellifera, are one of four big, world bees.It is suitble to Xinjiang is survived, but due to limited amount, endangered, is put into protection animal, therefore with higher economy and scientific research Value.The black bee in Xinjiang be distributed mainly on Xinjiang Yili of China, Tacheng, Altay, Xin Yuan, Tekes, Nilka, come to life, Gongliu County, Yining With the ground such as Burqin.Its primary raw material be the free of contamination fresh flower juice in height above sea level 1800m-2500m Tianshan Mountains depths, Major Nutrient into It is divided into minerals, organic acid, protein, vitamin and enzyme, refresh the mind protection stomach, the work(for the Antialcoholic liver-protecting that moistens the lung and relieve the cough Effect.

Royal jelly (Royal Jelly) also known as bee milk, abbreviation royal jelly are commonly called as queen bee breast, are a kind of milkys or yellowish The slurry of color, taste is sour and astringent and it is pungent to carry, slightly sweet taste.Root is studies show that, the ingredient of fresh royal jelly is quite multiple It is miscellaneous, wherein containing moisture 60%-70%, carbohydrate 10%-16%, protein 12%-15%, lipid 3%-6%, vitamin, trip The 2%-3% such as isolated amino acid and minerals.Wherein protein, carbohydrate are main component, and protein content is extremely abundant, accounts for about bee The 50% of royal jelly dry matter.There are many kinds of Nutrition and health functions for royal jelly, especially have very strong oxidation resistance, wherein Protein component be slow down aging key substance.Studies show that, royal jelly has the multinomial physical signs that can improve patient, Immunological regulation can be such as carried out, human blood-pressure is adjusted, reduces internal blood glucose, body cell is promoted to increase, also has antibacterial, is anti-inflammatory, anti- Tumour and other effects can also effectively reduce damages of the chemotherapeutics sub-chroniccisplatn to kidney.

Just in recent years to the research tendency of protein in royal jelly from the point of view of, explored gradually deeply from its basic function To the functional group to it and the discovery of structure and research.As royal jelly quality controling research, chemical constituent research, biology are lived Journal of Sex Research and its application etc..Bacteriostatic activity in royal jelly is just measured early in the 1960s, but does not know this antibacterial work Property it is whether related with wherein protein, therefore it is existing globulin in royal jelly is extracted, survey its bacteriostatic activity, finally determine bee Whether the globulin in royal jelly is antibacterial primary influence substance.

Completeness protein in protein and meat in royal jelly is a difference in that：Without neutral fat, not only Also containing 12 kinds of nonessential amino acid, therefore it is the good food of mankind's nourishing containing 10 kinds of essential amino acids.Free radical aging Theory thinks that the aging of the mankind is because human body generates and accumulates free radical too much, only removes these excessive free radicals, Healthy ability is secure.

For a long time, in order to preferably utilize protein, many researchers are dedicated to grinding for protolysate-polypeptide Study carefully.Protein can not only improve the digestibility of protein after protease moderately hydrolysis, keep and improve nutritive value, and And the functional characteristics such as its dissolubility, emulsibility, foaming characteristic and retentiveness can be improved.In addition, protein generated after enzymolysis certain A little low molecule peptide matters can not only provide growth in humans, the nutriment needed for development, and be more easy to be digested by human body, The effect of absorbing, while also there is diseases prevention, adjust human physiological functions.Low molecule peptide matters are as a kind of functional food base Material is obtained in all various aspects such as medicine, aerospace food, beverage, sports food, old people food and diet food and is widely answered With.At present, has the listing of commercialization polypeptide based food in West Europe, Japan and various countries of the U.S..The research phase of China's polypeptide based food To lag, in the starting stage.Studies in China it is more be soybean protein and lactalbumin, and to the research of royal jelly albumen still Without report.At present the research and deduction that are confined to its overall composition, research method are studied for royal jelly is antioxidative Also it is relatively simple.

Invention content

Present invention aims at provide a kind of preparation side of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang Method.

In order to achieve the above objectives, the present invention uses following technical proposals：The black bee royal jelly globulin in antioxidant activity Xinjiang The preparation method of enzymolysis product, includes the following steps：

(1) the black bee royal jelly freeze-dried powder in Xinjiang is prepared；

(2) degreasing and the main albumen of royal jelly is prepared；

(3) globulin is prepared；

(4) the black bee royal jelly globulin enzymolysis product in Xinjiang is prepared；

(5) different molecular weight peptide fragment in globulin enzymolysis product is detached.

The preparation method of the above-mentioned black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang, the Xinjiang in step (1) Black bee royal jelly freeze-dried powder preparation method is as follows：

Fresh black bee royal jelly is taken, after being weighed with assay balance, is put into centrifuge tube, then will be placed with fresh black bee queen bee The centrifuge tube of slurry is placed in -75~-85 DEG C of refrigerators, cold with vacuum freeze drier after fresh black bee royal jelly fully charge It is lyophilized dry to get the black bee royal jelly freeze-dried powder in Xinjiang.

The preparation method of the above-mentioned black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang in step (2), uses Following method degreasing：By the black bee royal jelly freeze-dried powder petroleum ether in Xinjiang temperature under conditions of 3~5 DEG C, with heat collecting type perseverance Warm 1~3h of magnetic stirrer, carries out degreasing, and filtering discards filtrate and obtains filter residue, then by filter residue petroleum ether in similarity condition Lower to repeat degreasing three times, last obtained filter residue is degreasing royal jelly.

The preparation method of the above-mentioned black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang in step (2), is prepared The method of the main albumen of royal jelly is as follows：

Degreasing royal jelly is dissolved in distilled water and extracts, filter, then filter residue distilled water is repeated to soak under similarity condition It carries three times, is then combined with filtrate, gained filtrate is royal jelly water solubility total protein solution, by royal jelly water solubility total protein solution After being dialysed with 14K dialysis membranes, reservation liquid is put into -75~-85 DEG C of refrigerators and is freezed, is freezed with vacuum freeze drier dry Up to the main protein freeze-dried powder of royal jelly after dry；Filter residue prepares raw material for globulin.

The preparation method of the above-mentioned black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang in step (2), uses One-step method degreasing and the water-soluble total protein of preparation, are as follows：

Bis- (2- ethoxys) amino (trihydroxy methyl) methane and N, N- (2- ethoxys) -2- ammonia are sequentially added in distilled water Base ethanesulfonic acid, makes a concentration of 0.3~0.5g/L, the N of bis- (2- ethoxys) amino (trihydroxy methyl) methane, and N- (2- ethoxys)- A concentration of 0.1~0.2g/L of 2-aminoethanesulfonic acid, stirring, the pH for adjusting solution are 7 to get royal jelly activation extraction liquid；So Backward royal jelly activation extraction liquid adds in the black bee royal jelly freeze-dried powder in Xinjiang, and 0.5~1h of stir-activating adds petroleum ether, often It rises royal jelly activation extraction liquid and adds in Xinjiang 4~7g of black bee royal jelly freeze-dried powder, the body of petroleum ether and royal jelly activation extraction liquid Product is than being 1:1.5~2；0.5~1h, filtering are vibrated, filter residue prepares raw material for globulin；Filtrate stratification removes organic phase； After water phase is dialysed with 14K dialysis membranes, reservation liquid is put into -75~-85 DEG C of refrigerators and is freezed, uses vacuum freeze drier Up to the main protein freeze-dried powder of royal jelly after freeze-drying.

The preparation method of the above-mentioned black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang, in step (3), in temperature Spend be 4 DEG C under conditions of, by degreasing and prepare 8~12 times of volumes of the filter residue after the main albumen of royal jelly, it is a concentration of 1.5~ The NaCl solution dissolving of 2.5wt% stirs 1.5~2h, filtering, by filter residue with a concentration of with heat collecting type constant temperature blender with magnetic force The NaCl solution of 1.5~2.5wt% repeats extraction three times under similarity condition, is dialysed, will retained with dialysis membrane after merging filtrate Liquid is put into freeze in -75~-85 DEG C of refrigerators after, after being freeze-dried with vacuum freeze drier gained dry powder be globulin.

The preparation method of the above-mentioned black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang, in step (3), in temperature Spend be 4 DEG C under conditions of, by degreasing and prepare the filter residue after the main albumen of royal jelly 8~12 times of volumes, NaCl concentration 1.5 The solution dissolving of a concentration of 0.3~0.6wt% of~2.5wt%, 4- hydroxyethyl piperazineethanesulfonic acids, pH=7, with heat collecting type constant temperature magnetic Power blender stirs 0.5~1h, and filtering dialyses filtrate with dialysis membrane, and reservation liquid is put into -75~-85 DEG C of refrigerators and is freezed Afterwards, gained dry powder is globulin after vacuum freeze drier freeze-drying.

The preparation method of the above-mentioned black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang, in step (4), weighs After 0.2500g globulin is dissolved in 62.5mL ultra-pure waters, it is divided into five parts：

First part is then placed in 37 DEG C of constant temperature convolutions with precision acidity meter tune pH to 2.0, addition 0.0667g pepsins 4h is shaken in oscillator or is put into 2h in globulin batch enzymolysis experimental provision；

Second part is then placed in 37 DEG C of constant temperature convolutions with precision acidity meter tune pH to 7.5, addition 0.0333g trypsase 4h is shaken in oscillator or is put into 2h in globulin batch enzymolysis experimental provision；

Third part is then placed in 37 DEG C of constant temperature and is returned with precision acidity meter tune pH to 8.5, addition 0.0333g coagulation protease trypsins 4h is shaken in rotation oscillator or is put into 2h in globulin batch enzymolysis experimental provision；

4th part is first adjusted to pH to 2.0 with precision acidity meter, adds in 0.0667g pepsins, is put into 37 DEG C of constant temperature convolutions Concussion 2h or 1h in globulin batch enzymolysis experimental provision is put into oscillator, then again with precision acidity meter tune pH to 7.5, 0.0333g trypsase is added in, be put into concussion 2h in 37 DEG C of constant temperature cyclotron oscillation devices or is put into globulin batch enzymolysis experiment 1h in device；

5th part, first with precision acidity meter tune pH to 2.0, adds in 0.1000g pepsins, is put into 37 DEG C of constant temperature convolutions and shakes It swings concussion 2h in device or is put into 1h in globulin batch enzymolysis experimental provision, later with precision acidity meter tune pH to 7.5, then add Enter 0.1000g trypsase, be put into concussion 2h in 37 DEG C of constant temperature cyclotron oscillation devices or be put into globulin batch enzymolysis experiment dress Interior 1h is put, then pH is adjusted to 8.5 with precision acidity meter, is eventually adding 0.1000g coagulation protease trypsins, 37 DEG C of constant temperature convolutions is put into and shakes It swings concussion 2h in device or is put into 1h in globulin batch enzymolysis experimental provision；

Above-mentioned five parts of solution is positioned over boiling water bath 15min in digital display type thermostat water bath after enzymolysis, inactivates enzyme； Centrifuge is used respectively, and centrifugal force 12000xg obtains enzymolysis product after centrifuging 10min at 4 DEG C：After first part of centrifugation It obtains obtaining enzymolysis product after enzymolysis product is named as pepsin enzymolysis product, second part of centrifugation and being named as trypsin digestion It obtains obtaining enzymolysis production after enzymolysis product is named as coagulation protease trypsin enzymolysis product, the 4th part of centrifugation after product, the centrifugation of third part Object obtains enzymolysis product and is named as stomach+pancreas+coagulation protease trypsin enzyme after being named as stomach+trypsin digestion product, the 5th part of centrifugation Solve product；

By enzymolysis product pepsin enzymolysis product obtained above, trypsin digestion product, coagulation protease trypsin enzymolysis Product, stomach+trypsin digestion product and stomach+pancreas+coagulation protease trypsin enzymolysis product, are 10kD and 3kD with molecular cut off Super filter tube or the royal jelly enzymolysis product ultrafiltration apparatus that molecular cut off is 10kD and 3kD carry out ultra-filtration and separation and obtain being more than 10kD Peptide fragment, 3kD-10kD peptide fragment and peptide fragment less than 3kD.

Beneficial effects of the present invention are as follows：

1st, the amount of globulin extracted from the black bee royal jelly in Xinjiang accounts for 1/3 or so of royal jelly dry weight, with expected results It is consistent.

2nd, royal jelly globulin enzymolysis product is obtained, and its inoxidizability is studied；Enzymolysis product resists Oxidation activity is more than non-enzymolysis product, best to the Scavenging activity of DPPH for coagulation protease trypsin in preceding four kinds of enzymolysis products, Secondly it is pepsin+trypsase, trypsase, most unstable is pepsin.Intersect enzymolysis by three kinds of protease Royal jelly globulin later can opposite increase to the elimination ability of DPPH free radicals.

3rd, with the antioxidant activity that the survey antioxidant activity measurement of Linoleic Acid Oxidation method is inhibited to determine enzymolysis product.First four kinds In the enzymolysis solution product that mode digests, inhibiting rate highest, that is, antioxidant activity of stomach+trypsin digestion product is most strong, is secondly Coagulation protease trypsin enzymolysis product antioxidant activity, trypsin digestion product antioxidant activity, the antioxygen of pepsin zymolyte Change ability is most weak.

4th, antioxidant activity has been carried out to three peptide fragments of enzymolysis product to be studied, antioxidant activity it is most strong be small In the peptide matters of 3kD, secondly the peptide matters of 3kD-10kD, the antioxidant activity more than 10kD segments is very weak.Royal jelly The substance that antioxidation is played in globulin is mainly the peptides less than 3kD.

5th, globulin extraction effect is determined using dying method with coomassie brilliant blue, carrying in globulin extraction process Take rate very high, the globulin purity in gained globulin is very high.

6th, the relationship of globulin degree of hydrolysis and oxidation resistance is studied, the enzymolysis degree highest at 7%-8%, Oxidation resistance is most strong.

Description of the drawings

The specific embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.

Fig. 1 differences enzymolysis product is to the Scavenging activity of DPPH；

(protein concentration of all peptide fragments is diluted to Scavenging activity of the enzymolysis product of Fig. 2 difference peptide fragments to DPPH 0.01mg/mL)；

The enzymolysis product total antioxidant capacity of Fig. 3 difference peptide fragments；

Fig. 4 is the structure diagram of globulin batch enzymolysis experimental provision；

Fig. 5 is the structure diagram that the level-one of globulin batch enzymolysis experimental provision digests the finned tube section of pipeline.

In Fig. 4 and Fig. 5：1-1- globulin storing containers；1-2- enzymolysis protease storing containers；1-3- housings；1-4- Level-one mixing vessel；1-5- level-ones digest pipeline；1-6- level-ones digest container；1-7- two level mixing vessels；1-8- secondary enzymolysis Pipeline；1-9- secondary enzymolysis containers；1-10- mixing agitating paddles；1-11- enzymolysis agitating paddles；1-12- mixing motors；1- 13- enzymolysis motors；1-14- micropumps；1-15- temperature sensors；1-16- heating plants；1-17- globulin enzymolysis products are deposited Put container；1-18- spoilers.

Fig. 6 is the structure diagram of globulin enzymolysis product batch ultrafiltration apparatus；

Fig. 7 is the structure diagram of the mixed flow tube of globulin enzymolysis product batch ultrafiltration apparatus.

In Fig. 6 and Fig. 7,1- enzymolysis product storing containers；The first solution feed pumps of 2-；The first polysulfone hollow fiber ultrafiltration membrane systems of 3-； 4- the first peritoneal effluent storage containers；5- the first concentrate cut-back tanks；6- the first rolling hyperfiltration membrane assemblies；The first flow-limiting valves of 7-； The second solution feed pumps of 8-；9- first distills water feeder；The first mixed flow tubes of 10-；11- spoilers；12- third solution feed pumps；13- Two polysulfone hollow fiber ultrafiltration membrane systems；14- the second peritoneal effluent storage containers；15- the second concentrate cut-back tanks；The second rollings of 16- Hyperfiltration membrane assembly；The second flow-limiting valves of 17-；The 4th solution feed pumps of 18-；19- after-fractionating water feeders；The second mixed flow tubes of 20-； 22- is more than 10kD peptide fragment storing containers；The peptide fragment storing containers of 23-3kD-10kD.

Specific embodiment

In order to illustrate more clearly of the present invention, the present invention is done further with reference to preferred embodiments and drawings It is bright.Similar component is indicated with identical reference numeral in attached drawing.It will be appreciated by those skilled in the art that institute is specific below The content of description is illustrative and be not restrictive, and should not be limited the scope of the invention with this.

1 materials and methods

1.1 material and reagent

Material：The black bee royal jelly in Xinjiang.

Reagent：Petroleum ether；NaCl；75wt% ethyl alcohol (AR)；NaOH(AR)；Diphenyl -2- hardship diazanyl free radicals (DPPH Free radical)；The phosphoric acid of 85wt%；Bovine serum albumin(BSA) (crystallization)；Formaldehyde (Tianjin Zhi Yuan chemical reagent Co., Ltd)；Tryptose Enzyme (Gaede chemical industry Tokyo compound probability Co., Ltd.)；Pepsin (Gaede chemical industry Tokyo compound probability Co., Ltd.)；Pancreas coagulates Protease (Gaede chemical industry Tokyo compound probability Co., Ltd.)；Ferric ferricyanide；Ferric trichloride；Anhydrous ferric chloride (Shandong West Asia Learn Industrial Co., Ltd)；Coomassie brilliant blue G -250 (Sigma companies)；Linoleic acid；Total antioxidation reagent (Beijing Suo Laibaoke Skill Co., Ltd)；Phosphate buffer (Shandong West Asia limited company)；Ammonium thiocyanate etc..

1.2 laboratory apparatus

Centrifuge tube；Super filter tube or enzymolysis product ultrafiltration apparatus (self-control)；Beaker；Test tube；Rack for test tube；Pipette；14K dialyses Film；FD-1A-50 vacuum freeze driers (gather same Electronics Co., Ltd. in Hangzhou)；HJ8 heat collecting type constant temperature blender with magnetic force (Guangzhou Hu Ruiming Instrument Ltd.)；FA1004B electronic balances (Zhengzhou Bao Jing Electronic Science and Technology Co., Ltd.s)；TD5Z at a high speed freeze from Scheming (Shanghai Co., Ltd of Zhao Di biologies section)；HH.S21-4 digital display types thermostat water bath (Xingtai profit connection limited public affairs of mechanical equipment Department)；SHA-B constant temperature oscillators (Jintan City of Jiangsu Province You Lian instruments research institute) or globulin batch enzymolysis experimental provision are (certainly System)；D-9143B-1 electric heating constant-temperature blowing drying boxes (remittance that instrument)；(Beijing is put down builds reality to TGL-16G high speed tabletop centrifuges Yan Shi equipment Co., Ltd)；KQ-100B ultrasonic cleaners (Shanghai City ultrasonic instrument Co., Ltd)；UV2400 spectrophotometrics It counts (Shun's space perseverance is put down)；PHS-3C Accurate pHs adjust meter (upper marine rainbow benefit instrument and meter Co., Ltd., Factory).

2 experimental methods

2.1. in the black bee royal jelly in Xinjiang globulin preparation；

Fresh black bee royal jelly is taken first, after being weighed with assay balance, is placed in -80 DEG C of refrigerators, treats fresh black bee queen bee After starching fully charge, after being freeze-dried with vacuum freeze drier, dry powder is obtained.

Degreasing：Gained dry powder petroleum ether is in temperature to be stirred under conditions of 4 DEG C, with heat collecting type constant temperature blender with magnetic force 2h, carries out degreasing, and filtering discards filtrate and obtains filter residue, then filter residue petroleum ether is repeated degreasing three times under similarity condition, finally Obtained filter residue is degreasing royal jelly.

It prepares water-soluble total protein and obtains globulin and prepare raw material：The degreasing royal jelly of gained is dissolved in distilled water and is soaked It carries, filters, then filter residue distilled water is repeated into extraction three times under similarity condition, be then combined with filtrate, gained filtrate is queen bee Pulp-water dissolubility total protein solution；Filter residue prepares raw material for globulin.

In order to simplify the flow and process that degreasing, the water-soluble total protein of preparation and acquisition globulin prepare raw material, water is improved The yield of dissolubility total protein reduces impurity and provides following one-step method degreasing and system to subsequently preparing the influence of globulin, the present invention Standby water solubility total protein and acquisition globulin prepare raw material：Bis- (2- ethoxys) amino (three hydroxyl first are sequentially added in distilled water Base) methane and N, N- (2- ethoxys) -2-aminoethanesulfonic acid, make a concentration of of bis- (2- ethoxys) amino (trihydroxy methyl) methane 0.3g/L, N, a concentration of 0.1g/L of N- (2- ethoxys) -2-aminoethanesulfonic acid, stirring, the pH for adjusting solution are 7 to get bee Royal jelly activation extraction liquid；Then to royal jelly activation extraction liquid add in the black bee royal jelly freeze-dried powder in Xinjiang, stir-activating 0.5h, then Petroleum ether is added in, every liter of royal jelly activation extraction liquid adds in the black bee royal jelly freeze-dried powder 6g in Xinjiang, and petroleum ether is activated with royal jelly The volume ratio of extracting solution is 1:1.5；0.5h, filtering are vibrated, filter residue prepares raw material for globulin；Filtrate stratification, removing have Machine phase, water phase are royal jelly water solubility total protein solution.One-step method degreasing and the water-soluble total protein of preparation and acquisition globulin Raw material is prepared with following technological merit：(1) reduce the number of degreasing operation：Adding in after petroleum ether only needs once can To realize the complete degreasing of royal jelly, gained filter residue with petroleum ether is extracted again, is also failed in petroleum ether using liquid chromatogram Detect lipid material, this explanation is by the thorough degreasing of dry powder；And simply using petroleum ether degreasing, it, will after degreasing five times Gained filter residue still can detect lipid material with petroleum ether extraction in petroleum ether again.(2) it is water-soluble to reduce preparation Extracting times during total protein：Water is carried to carry with ether and is combined into one, not only simplifies experimental procedure, and by once extracting it Afterwards, again with flooding filter residue is distilled, having can't detect protein in filtrate, (specific detection method is referring to GB9697- 2008), this illustrates that the extraction of water-solubility protein is very thorough；If extracted using distilled water merely, at least need to carry out six times Above extraction could be realized and can't detect protein in filtrate.

Royal jelly water solubility total protein solution with the dialysis membrane that aperture is 14K is dialysed, liquid will be retained and be put into -80 DEG C It in refrigerator, after its fully charge, is freeze-dried with vacuum freeze drier, gained dry powder is the main albumen of royal jelly； Royal jelly water solubility total protein solution is put into -80 DEG C of refrigerators, after its fully charge, is carried out with vacuum freeze drier cold It is lyophilized dry, gained water solubility total protein freeze-dried powder is dissolved in ultra-pure water, and it is 6.0mg/mL's to be configured to water-soluble total protein concentration Royal jelly water solubility total protein solution heats 10min, then with centrifuge at 100 DEG C with thermostat water bath, removes queen bee Main albumen is starched, after supernatant liquor is taken to be put into and freeze in -80 DEG C of refrigerators, is lyophilized with vacuum freeze drier, obtains other water solubilitys Albumen.

Under conditions of temperature is 4 DEG C, globulin is prepared into NaCl solution of the raw material with a concentration of 2wt% of 10 times of volumes Dissolving stirs 2h, filtering, by filter residue with the NaCl solution of a concentration of 2wt% in same batten with heat collecting type constant temperature blender with magnetic force Extraction is repeated under part three times, is dialysed after merging filtrate with dialysis membrane, is retained after liquid is put into and freezes in -80 DEG C of refrigerators, it is cold with vacuum Gained dry powder is globulin after lyophilizer freeze-drying.

In order to improve the extraction efficiency of globulin, simplify extraction step, using 10 times of volumes, NaCl concentration 2wt%, The solution dissolving globulin of 4- hydroxyethyl piperazineethanesulfonic acids a concentration of 0.4wt%, pH=7 prepare raw material, with heat collecting type constant temperature magnetic Power blender stirs 2h, and filtering dialyses filtrate with dialysis membrane, reservation liquid is put into after freezing in -80 DEG C of refrigerators, cold with vacuum Gained dry powder is that globulin (repeats to extract, egg in dry powder compared to a concentration of 2wt%NaCl after lyophilizer freeze-drying 10%) white matter purity improves.Filter residue is extracted again with the NaCl solution of a concentration of 2wt%, using in the prior art Dying method with coomassie brilliant blue survey filtrate in globulin content, can't detect globulin, globulin recovery rate approaches 100%.And repeat to extract three times and then secondary extraction filter residue using the NaCl solution of a concentration of 2wt% merely, use existing skill Dying method with coomassie brilliant blue in art surveys globulin content in filtrate, is still able to detect that globulin, repeats extraction 10 and takes second place It can not just detect afterwards.

2.2. in the black bee royal jelly in Xinjiang globulin enzymolysis product preparation；

It weighs after 0.2500g globulin is dissolved in 62.5mL ultra-pure waters, is divided into five parts：

Above-mentioned five parts of solution is positioned over boiling water bath 15min in digital display type thermostat water bath after enzymolysis, inactivates enzyme； Centrifuge is used respectively, and centrifugal force 12000xg obtains enzymolysis product after centrifuging 10min at 4 DEG C：After first part of centrifugation It obtains obtaining enzymolysis product after enzymolysis product is named as pepsin enzymolysis product, second part of centrifugation and being named as trypsin digestion It obtains obtaining enzymolysis production after enzymolysis product is named as coagulation protease trypsin enzymolysis product, the 4th part of centrifugation after product, the centrifugation of third part Object obtains enzymolysis product and is named as stomach+pancreas+coagulation protease trypsin enzyme after being named as stomach+trypsin digestion product, the 5th part of centrifugation Solve product.

As shown in Figure 4 and Figure 5, globulin batch enzymolysis includes globulin storing containers 1-1, globulin enzyme with experimental provision Solve container, micropump 1-14, enzymolysis protease storing containers 1-2 and globulin enzymolysis product storing containers 1-17, the ball Proteolysis container includes housing 1-3, level-one mixing vessel 1-4, level-one enzymolysis pipeline 1-5, level-one enzymolysis container 1-6, two level Incubation cavity, the level-one are equipped in mixing vessel 1-7, secondary enzymolysis pipeline 1-8 and secondary enzymolysis container 1-9, the housing 1-3 It is mixing vessel 1-4, level-one enzymolysis pipeline 1-5, level-one enzymolysis container 1-6, the two level mixing vessel 1-7, described Secondary enzymolysis pipeline 1-8 and the secondary enzymolysis container 1-9 are separately positioned in the incubation cavity；The globulin storing containers The outlet end of 1-1 and the enzymolysis with the outlet end of protease storing containers 1-2 respectively with the level-one mixing vessel 1-4 into The fluid communication connection of liquid end, the liquid feeding end of the outlet end and level-one enzymolysis container 1-6 of the level-one mixing vessel 1-4 pass through The level-one enzymolysis pipeline 1-5 fluid communication connections, the outlet end of the level-one enzymolysis container 1-6 is with the micropump 1-14's Liquid feeding end fluid communication connects, and the outlet end of the micropump 1-14 and the liquid feeding end fluid of the two level mixing vessel 1-7 are led Leading to and connect, the outlet end of the two level mixing vessel 1-7 is connect with the liquid feeding end fluid communication of the secondary enzymolysis container 1-9, The outlet end of the secondary enzymolysis container 1-9 is connect with the globulin enzymolysis product storing containers 1-17 fluid communications；It is described The level-one enzymolysis pipeline 1-5 and secondary enzymolysis pipeline 1-8 is the finned tube pipeline that inner wall is equipped with spoiler 1-18；It is described The high temperature medium import of incubation cavity is connect with the high temperature media outlet fluid communication of heating plant 1-16.The level-one digests pipeline The 1-5 and secondary enzymolysis pipeline 1-8 is spiral pipeline, and high temperature medium is hot-air.In order to improve globulin, protease with And mixture homogeneity of the water in the level-one mixing vessel 1-4 and in the two level mixing vessel 1-7, it is mixed in the level-one Be provided with mixing agitating paddle 1-10 in container 1-4 and in the two level mixing vessel 1-7, the mixing with agitating paddle 1-10 with Mixing is sequentially connected, while with the output shaft of motor 1-12 for the uniformity for improving globulin enzyme digestion reaction, also described one Enzymolysis agitating paddle 1-11, the enzymolysis agitating paddle are equipped in grade enzymolysis container 1-6 and in the secondary enzymolysis container 1-9 1-11 is sequentially connected with the enzymolysis output shaft of motor 1-13.In view of protease can inactivate under the influence of extraneous factor, because This needs in the middle part of enzyme digestion reaction carry out process to supplement protease to enzyme digestion reaction system, can also will disposably add originally Albumen enzyme amount adds in two times, therefore the enzymolysis outlet end of protease storing containers 1-2 is mixed appearance with the two level The liquid feeding end fluid communication connection of device 1-7.And in view of the temperature of the activity and reaction system of enzyme has certain relationship, in order to keep away Exempt from that local temperature is excessively high or local temperature is too low, set on level-one enzymolysis pipeline 1-5 there are three temperature sensor 1-15, On level-one enzymolysis pipeline 1-5, first temperature sensor 1-15 is arranged on the feed liquor of the level-one enzymolysis pipeline 1-5 Mouthful, second temperature sensor 1-15 is arranged in the pipe of the level-one enzymolysis pipeline 1-5 terminals, third temperature sensor 1- 15 are arranged on the liquid outlet of level-one enzymolysis pipeline 1-5, and set on the secondary enzymolysis pipeline 1-8 that there are three temperature sensing Device 1-15, on the secondary enzymolysis pipeline 1-8, first temperature sensor 1-15 is arranged on the secondary enzymolysis pipeline 1-8 Inlet, second temperature sensor 1-15 be arranged in the pipe of the secondary enzymolysis pipeline 1-8 terminals, and third temperature passes Sensor 1-15 is arranged on the liquid outlet of the secondary enzymolysis pipeline 1-8, while temperature sensor 1- is equipped in the incubation cavity 15.When carrying out globulin enzyme digestion reaction, the globulin of the globulin storing containers 1-1 is added in into the level-one mixing vessel 1- In 4, and the enzymolysis is added to the level-one mixing vessel 1-4 with a part of protease in protease storing containers 1-2 It is interior, and the mixing in the level-one mixing vessel 1-4 with agitating paddle 1-10 under the action of mixed, after preliminary mixing Globulin, protease and enzyme digestion reaction distilled water digest pipeline 1-5 along the level-one and digest container to the level-one 1-6 flows, and in flow process, under the flow-disturbing effect of the spoiler 1-18 further mixing occurs for mixed solution, together When globulin tentatively digested under the catalytic action of protease, the mixed solution that is tentatively digested flows into the level-one enzyme After solving in container 1-6, enzymolysis agitating paddle 1-11 stirring action of the solution to be mixed in level-one enzymolysis container 1-6 Under after reaction a period of time, recycle the micropump 1-14 that mixed solution is pumped into institute in level-one enzymolysis container 1-6 It states in two level mixing vessel 1-7, while by the enzymolysis described in a part of protease addition in protease storing containers 1-2 In two level mixing vessel 1-7, under the action of then the mixing in the two level mixing vessel 1-7 is with agitating paddle 1-10 into Row mixing, mixed solution is flowed along the secondary enzymolysis pipeline 1-8 to the secondary enzymolysis container 1-9, in flow process In, further mixing, while the ball egg in mixed solution occur under the flow-disturbing effect of the spoiler 1-18 for mixed solution Further enzyme digestion reaction can be carried out under the catalytic action of protease in vain, mixed solution flows into the secondary enzymolysis container 1-9 Afterwards, the enzymolysis in the secondary enzymolysis container 1-9 is under agitating paddle 1-11 stirring actions, the globulin in mixed solution Continue enzyme digestion reaction, treat after reaction a period of time can reaction solution be discharged into globulin enzymolysis product storage and hold It is preserved in device 1-17.Mixed solution can be not only improved using the level-one enzymolysis pipeline 1-5 and secondary enzymolysis pipeline 1-8 Mixability and the uniformity that is distributed in mixed solution of protease, enzyme digestion reaction path can also be extended, while also just In being heated and kept the temperature to reaction solution, so as to so that the Degree of Enzymatic Hydrolysis of globulin obtains in reaction time when identical To effectively improving.

2.3. in globulin enzymolysis product different molecular weight peptide fragment preparation；

Enzymolysis product pepsin enzymolysis product, trypsin digestion product, the coagulation protease trypsin that will be obtained in step 2.2 Enzymolysis product, stomach+trypsin digestion product and stomach+pancreas+coagulation protease trypsin enzymolysis product, are respectively 10kD with molecular cut off The globulin enzymolysis product ultrafiltration apparatus that super filter tube or molecular cut off with 3kD are 10kD and 3kD carries out ultra-filtration and separation and obtains The peptide fragment of peptide fragment, 3kD-10kD more than 10kD and the peptide fragment less than 3kD.

As shown in Figure 6 and Figure 7, globulin enzymolysis product batch ultrafiltration apparatus include more than 10kD peptides separation system and The peptides separation system of 3kD-10kD, the peptides separation system more than 10kD include globulin enzymolysis product storing containers 1, first Solution feed pump 2, the first polysulfone hollow fiber ultrafiltration membrane system 3, the first concentrate cut-back tank 5, the second solution feed pump 8, the first roll-to-roll ultrafiltration 6 and first peritoneal effluent storage container 4 of membrane module, the outlet end of the globulin enzymolysis product storing containers 1 are supplied with described first The liquid feeding end fluid communication connection of liquid pump 2, the outlet end of first solution feed pump 2 and first polysulfone hollow fiber ultrafiltration membrane system 3 liquid feeding end fluid communication connection, the concentrate outlet end of first polysulfone hollow fiber ultrafiltration membrane system 3 and the described first concentration The liquid feeding end fluid communication connection of liquid cut-back tank 5, outlet end and second feed flow of the first concentrate cut-back tank 5 The liquid feeding end fluid communication connection of pump 8, the outlet end of second solution feed pump 8 and the first rolling hyperfiltration membrane assembly 6 into The fluid communication connection of liquid end, the peritoneal effluent outlet end of the first rolling hyperfiltration membrane assembly 6 and the feed liquor of first solution feed pump 2 Hold fluid communication connection；The peritoneal effluent outlet end of first polysulfone hollow fiber ultrafiltration membrane system 3 is stored with first peritoneal effluent The liquid feeding end fluid communication connection of container 4；The concentrate outlet end of the first rolling hyperfiltration membrane assembly 6 is with being more than 10kD peptide fragments The liquid feeding end fluid communication connection of storing containers 22.Wherein, in order to avoid first solution feed pump 2 is to first doughnut Large effect is generated to ultrafiltration system in the first rolling hyperfiltration membrane assembly 6 during pumping liquid in hyperfiltration membrane assembly 3, in institute The peritoneal effluent outlet end for stating the first rolling hyperfiltration membrane assembly 6 is provided with the first flow-limiting valve 7, is limited by first flow-limiting valve 7 The flow of 2 liquid feeding end of the first solution feed pump is flowed to by the peritoneal effluent outlet end of the first rolling hyperfiltration membrane assembly 6.And in order to The concentrate flowed out from the concentrate outflow end of first polysulfone hollow fiber ultrafiltration membrane system 3 is improved to be uniformly mixed with distilled water Degree, by the concentrate outlet end of first polysulfone hollow fiber ultrafiltration membrane system 3 and the feed liquor of the first concentrate cut-back tank 5 End is connected by 10 fluid communication of the first mixed flow tube, and first mixed flow tube 10 is equipped with the diversion pipe of spoiler 11 for inner wall, The liquid feeding end of first mixed flow tube 10 is connect, and described the with the outlet end fluid communication of the first distillation water feeder 9 Agitating paddle, the output shaft drive connection of the agitating paddle and stirring motor are equipped in one concentrate cut-back tank 5.In use, it opens It moves first solution feed pump 2 globulin enzymolysis product is pumped into first polysulfone hollow fiber ultrafiltration membrane system 3, described first Under the centrifugation of polysulfone hollow fiber ultrafiltration membrane system 3, globulin enzymolysis product is divided into peritoneal effluent and concentrate, and peritoneal effluent is by pipe Road flows into the first peritoneal effluent storage container 4 and stores, and it is dense that concentrate then flows into described first via first mixed flow tube 10 In contracting liquid cut-back tank 5, when concentrate flows through first mixed flow tube 10, the spoiler 11 flow-disturbing effect under with it is described The distilled water mixing that first distillation water feeder 9 flows out, after flowing into the first concentrate cut-back tank 5, concentrate It can be sufficiently mixed under the stirring action of the agitating paddle with distilled water, so as to make the water-soluble substances in concentrate abundant Ground is dissolved in water, is pumped into the first rolling hyperfiltration membrane assembly 6 via second solution feed pump 8 by diluted concentrate, And detached by the first rolling hyperfiltration membrane assembly 6, it is carried out via the first rolling hyperfiltration membrane assembly 6 isolated Concentrate is directly discharged into more than in 10kD peptide fragments storing containers 22, and by the first rolling hyperfiltration membrane assembly 6 detach To peritoneal effluent be then pumped into first polysulfone hollow fiber ultrafiltration membrane system 3 by first solution feed pump 2 and detached again.Institute It states the first rolling hyperfiltration membrane assembly 6 and secondary separation is carried out to the concentrate after dilution, and will be by the first rolling ultrafiltration membrane group The isolated peritoneal effluent of part 6 is detached again, so as to which water-soluble substances in globulin enzymolysis product are as fast as possible It separates fastly, to provide sample to carry out multigroup characteristic and physiological function test to globulin enzymolysis product.

The peptides separation system of 3kD-10kD includes third solution feed pump 12, the second polysulfone hollow fiber ultrafiltration membrane system 13, second Concentrate cut-back tank 15, the 4th solution feed pump 18, the second rolling hyperfiltration membrane assembly 16 and the second peritoneal effluent storage container 14, it is described The outlet end of first peritoneal effluent storage container 4 is connect with the liquid feeding end fluid communication of second solution feed pump 12, and described second supplies The outlet end of liquid pump 12 is connect with the liquid feeding end fluid communication of second polysulfone hollow fiber ultrafiltration membrane system 13, and described second is hollow The concentrate outlet end of cellulosic ultrafiltration membrane module 13 is connect with the liquid feeding end fluid communication of the second concentrate cut-back tank 15, The outlet end of the second concentrate cut-back tank 15 is connect with the liquid feeding end fluid communication of the 4th solution feed pump 18, and described The outlet end of four solution feed pumps 18 is connect with the liquid feeding end fluid communication of the second rolling hyperfiltration membrane assembly 16, second rolling The peritoneal effluent outlet end of hyperfiltration membrane assembly 16 is connect with the liquid feeding end fluid communication of second solution feed pump 12；Described second is hollow The peritoneal effluent outlet end of cellulosic ultrafiltration membrane module 13 is connect with the liquid feeding end fluid communication of the second peritoneal effluent storage container 14； The liquid feeding end fluid of the concentrate outlet end of the second rolling hyperfiltration membrane assembly 16 and the peptide fragment storing containers 23 of 3kD-10kD Conducting connection.Wherein, in order to avoid the third solution feed pump 12 is pumped into liquid into second polysulfone hollow fiber ultrafiltration membrane system 13 Large effect is generated to ultrafiltration system in the second rolling hyperfiltration membrane assembly 16 during body, in the second rolling ultrafiltration membrane group The peritoneal effluent outlet end of part 16 is provided with the second flow-limiting valve 17, is limited by second flow-limiting valve 17 and is surpassed by second rolling The peritoneal effluent outlet end of filter membrane component 16 flows to the flow of 12 liquid feeding end of third solution feed pump.And in order to improve from described second The concentrate of concentrate outflow end outflow and distilled water mixture homogeneity of polysulfone hollow fiber ultrafiltration membrane system 13, will be in described second The concentrate outlet end of fibre ultrafiltration membrane module 13 and the liquid feeding end of the second concentrate cut-back tank 15 are mixed by second 20 fluid communication of flow tube connects, and second mixed flow tube 20 is equipped with the diversion pipe of spoiler 11, second mixed flow for inner wall The liquid feeding end of pipe 20 is connect with the outlet end fluid communication of after-fractionating water feeder 19, and second concentrate dilutes Agitating paddle, the output shaft drive connection of the agitating paddle and stirring motor are equipped in container 15.It is supplied in use, starting the third Globulin enzymolysis product is pumped into second polysulfone hollow fiber ultrafiltration membrane system 13 by liquid pump 12, is surpassed in second doughnut Under the centrifugation of filter membrane component 13, globulin enzymolysis product is divided into peritoneal effluent and concentrate, and peritoneal effluent flows into institute by pipeline It states the second peritoneal effluent storage container 14 to store (peptide fragment as less than 3kD), concentrate is then flowed via second mixed flow tube 20 Enter in the second concentrate cut-back tank 15, when concentrate flows through second mixed flow tube 20, in disturbing for the spoiler 11 The lower distilled water flowed out with the after-fractionating water feeder 19 of stream effect mixes, and flows into the second concentrate cut-back tank After in 15, concentrate and distilled water can be sufficiently mixed under the stirring action of the agitating paddle, so as to make in concentrate Water-soluble substances fully dissolve in water, be pumped into the volume Two via the 4th solution feed pump 18 by diluted concentrate It in formula hyperfiltration membrane assembly 16, and is detached by the second rolling hyperfiltration membrane assembly 16, via second rolling ultrafiltration membrane Component 16 carries out isolated concentrate and is directly discharged into the peptide fragment storing containers 23 of 3kD-10kD, and by second rolling Hyperfiltration membrane assembly 16 carries out isolated peritoneal effluent and is then pumped into second Hollow Fiber Ultrafiltration by the third solution feed pump 12 It is detached again in membrane module 13.The second rolling hyperfiltration membrane assembly 16 carries out secondary separation to the concentrate after dilution, And will again be detached by the isolated peritoneal effluent of the second rolling hyperfiltration membrane assembly 16, so as to by globulin enzyme Solution water in products soluble substance is separated as quickly as possible, so as to for globulin enzymolysis product is carried out multigroup characteristic and Physiological function test provides sample.

The peptides separation system of peptides separation system and 3kD-10kD more than 10kD is using double hyperfiltration membrane assemblies to ball Protein hydrolysate carries out separation and Extraction, not only can further reduce the content of water-soluble substances in concentrate, but also can To improve the efficiency of globulin enzymolysis product separation, it shorten to research globulin enzymolysis product characteristic and physiological function provides sample Time, so as to meet while the sample requirement of multigroup experiment.

2.4, which remove DPPH methods, surveys globulin zymolyte antioxidant activity (with reference to test method of the prior art)

Diphenyl -2- hardship diazanyls free radical (DPPH free radicals) Scavenging activity measures：Diphenyl -2- hardship diazanyl free radicals (DPPH free radicals) is a kind of free radical being well used, be dissolved in it is dark purple and extremely stable after ethyl alcohol, Lone electron pair has strong absorption near 516nm-518nm.When adding in the substance for removing DPPH free radicals, 1,1 '-phenyl-hardship The lone electron pair of diazanyl free radical (DPPH free radicals) is matched, and absorbance also and then weakens or disappears, and darkviolet becomes Lilac, its fading extent and the electronics that it receives keep certain proportionate relationship to quantity, make it at maximum wavelength Absorption values are reduced.Judge globulin zymolyte to 1,1 '-phenyl-hardship hydrazine by measuring the numerical value change of solution absorbance The Scavenging activity of base free radical (DPPH free radicals).

Absolute ethyl alcohol, which dissolves 1,1- phenyl-hardship diazanyl free radical (DPPH free radicals), makes its a concentration of 0.2mmol/L, will Sample to be tested is diluted with ultra-pure water by certain dilution gradient and is configured to testing sample solution, take testing sample solution 2mL with The DPPH free radical ethanol solution mixings of a concentration of 0.2mmol/L of 2mL using solvent as control, survey its extinction at 517nm It spends (Ai)；Testing sample solution 2mL is taken, the abundant mixing of 2mL absolute ethyl alcohols is added in, measures the absorbance (Aj) at 517nm；It surveys Determining placebo solution, (mixing of the DPPH free radical ethanol solutions of a concentration of 0.2mmol/L of 2mL ultra-pure waters and 2mL is molten Liquid) the absorbances (Ac) that go out of 517nm.

DPPH inhibiting rates (%)=1- [(Ai)-(Aj)]/Ac] } × 100%；

Ai：Absorbance of the sample at 517nm during using solvent as reference material；

Aj：Absorbance of the sample to be tested at 517nm；

Ac：Absorbance of the blank control at 517nm.

2.5 inhibit Linoleic Acid Oxidation method to survey antioxidant activity (with reference to test method of the prior art)

Each 1mL of black royal jelly globulin each component sample to be tested of prepared a concentration of 0.01mg/mL is taken, is separately added into 1mL volumetric concentrations are the linoleic acid ethanol solution of 2.50% (V/V), add the phosphate of a concentration of 0.05mol/L of 2mL Buffer solution (pH 7.0) and 1mL absolute ethyl alcohols are placed on dark place and 37 DEG C of constant temperature are kept to preserve after closed.Blank group is not added with antioxygen Agent, other steps are same as above.

The above-mentioned mixed liquors of 0.5mL are taken, the ethanol solution and 0.5mL mass fractions that addition 5.00mL volumetric concentrations are 75% are 30% NH₄SCN adds the FeCl of a concentration of 0.02mol/L of 0.5mL₂Hydrochloric acid solution (FeCl₂The preparation method of hydrochloric acid solution For：By FeCl₂It is added in the hydrochloric acid that mass fraction is 3.5% so that FeCl₂A concentration of 0.02mol/L), react 3min Afterwards, its absorbance at 500nm is surveyed, it is primary every measuring for 24 hours later.

2.6 total antioxidant capacity assay methods (with reference to test method of the prior art)

The black royal jelly globulin each component samples of 0.01mg/mL are prepared, illustrate to be operated according to T-AOC kits, Absorbance is surveyed at 520nm.Experiment is repeated 3 times, and takes its average value.This experiment is with sample cell and extinction of the control tube at 520nm The size of the difference of degree reacts its total antioxidant capacity, and difference is bigger, total antioxidant capacity is stronger.

2.7 dying method with coomassie brilliant blue survey globulin content (with reference to test method of the prior art)

Draw standard curve：

Compound concentration is the bovine serum albumin(BSA) standard of 20 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL and 100 μ g/mL Solution is reacted with Coomassie brilliant blue G -250, with absorbance A₅₉₅Nm, using standard protein content as abscissa, is painted as ordinate Standard curve processed acquires calibration curve equation (R²=0.9997).

Unknown sample determination of protein concentration：

Assay method is same as above, and gained globulin is configured to solution, according to the Ai of the 595nm measured, in standard curve On find it and be equivalent to the amount of standard protein, so as to calculate the protein concentration of unknown sample (μ g/mL).

The measure of 2.8 degree of hydrolysis (with reference to test method of the prior art)

Degree of hydrolysis is measured using formol titration, formol titration is according to-NH₃ ⁺It is combined with formaldehyde and forms-NH- CH₂OH、-N-(CH₂OH)₂Hydroxymethyl derivatives are waited, so as to make-NH₃ ⁺On H⁺It releases, uses alkalimetric titration separate out in this way H⁺, so as to calculate degree of hydrolysis.

3 interpretations of result

3.1DPPH removes method and surveys oxidation resistance result and analysis

As can be seen from Figure 1 zymolyte is to the Scavenging activity of DPPH：

When concentration is in 2-4 μ g/mL, pepsin enzymolysis product+trypsin digestion product ＞ coagulation protease trypsins enzymolysis Product ＞ trypsin digestion product ＞ pepsin enzymolysis products；

When concentration is more than 4 μ g/mL, coagulation protease trypsin enzymolysis product ＞ pepsins enzymolysis product+trypsin digestion Product ＞ trypsin digestion product ＞ pepsin enzymolysis products；

And inhibiting rate is from high to low in respective three peptide fragments of three bulb protein zymolytes：Less than 3kD ＞ 3kD-10kD ＞ is more than 10kD's.

In summary analysis is it is found that when concentration is in 2-4 μ g/mL, the common enzymolysis product of pepsin+trypsase it is clear Removing solid capacity is most strong；When concentration is more than 4 μ g/mL, the Scavenging activity of coagulation protease trypsin enzymolysis product is most strong；4- globulin during enzymolysis Zymolyte is most strong to the Scavenging activity of protein, as shown in Figure 2：Peptide fragment is smaller, and protein is stronger to the Scavenging activity of DPPH.

3.2 inhibit Linoleic Acid Oxidation method to survey antioxidant activity result and analysis

As shown in table 1, the enzymolysis product of royal jelly globulin has certain inhibiting effect, stomach egg to linoleic oxidation The inhibition of royal jelly globulin after white enzyme and the common enzymolysis of trypsase is most strong, the royal jelly ball egg after pepsin enzymolysis White inhibition is most weak.The strong and weak sequence of enzymolysis product anti-Linoleic Acid Oxidation effect is：The enzymolysis product of pepsin+trypsase The enzymolysis product of the enzymolysis product ＞ pepsins of the enzymolysis product ＞ trypsase of ＞ coagulation protease trypsins, this and remove above The strong and weak sequence consensus of DPPH.Royal jelly water solubility peptide fragment of the molecular weight less than 3kD inhibits the oxidation resistant ability of linoleic acid most By force.

Table 1：Add in the linoleic acid-NH of royal jelly globulin enzymolysis product₄SCN-FeCl₂Reaction system is inhaled afterwards

Luminosity changes with time

Sample	1	2	3	4	5
						Blank control	0.056	0.155	0.136	0.174	0.162
Globulin	0.087	0.182	0.269	0.345	0.427
						Stomach cardia enzymolysis product	0.120	0.090	0.144	0.154	0.157
Pepsin enzymolysis ＞ 10kD	0.048	0.064	0.075	0.109	0.161
						Pepsin digests 3-10kD	0.055	0.081	0.104	0.152	0.093
Pepsin enzymolysis ＜ 3kD	0.076	0.064	0.116	0.085	0.112
						Tryptose enzymolysis product	0.056	0.076	0.089	0.094	0.068
Trypsin digestion ＞ 10kD	0.044	0.065	0.080	0.092	0.042
						Trypsin digestion 3-10kD	0.056	0.078	0.086	0.114	0.125
Trypsin digestion ＜ 3kD	0.035	0.067	0.085	0.086	0.043
						Coagulation protease trypsin solution product	0.023	0.066	0.084	0.101	0.050
Coagulation protease trypsin enzymolysis ＞ 10kD	0.024	0.076	0.089	0.064	0.054
						Coagulation protease trypsin digests 3-10kD	0.023	0.067	0.105	0.098	0.103
Coagulation protease trypsin enzymolysis ＜ 3kD	0.055	0.038	0.087	0.084	0.075
						Pepsin+trypsin digestion product	0.045	0.067	0.083	0.108	0.087
Stomach+pancreatin solution ＞ 10kD	0.023	0.061	0.102	0.095	0.088
						Stomach+pancreatin solution 3-10kD	0.015	0.054	0.063	0.125	0.094
Stomach+pancreatin solution ＜ 3kD	0.013	0.036	0.059	0.093	0.087

The result of the measure of 3.3 total antioxidant capacity and analysis

As shown in Figure 3：The total antioxidation energy of the peptide fragment more than 10kD is separated to from the enzymolysis product of royal jelly globulin The equal very little of power, and the segment less than 3kD is respectively provided with very high total antioxidant capacity.The enzymolysis product total antioxidation effect of globulin Strong and weak sequence be：The enzyme of the enzymolysis product ＞ trypsase of the enzymolysis product ＞ coagulation protease trypsins of pepsin+trypsase Solve the enzymolysis product of product ＞ pepsins.Molecular weight is stronger less than the peptide fragment total antioxidant capacity of 3kD.

3.4 protein content determination results and analysis

By being measured to the globulin extracted, the content of the globulin in the black bee royal jelly in Xinjiang accounts for about royal jelly The 1/3 of dry weight.

3.5 degree of hydrolysis measurement results and analysis

Understand that its degree of hydrolysis is respectively 5.41%, 6.34%, 7.62%, 7.83%, 9.61% from 2 measured data of table, Measurement result is stablized relatively, it is known that the Degree of Enzymatic Hydrolysis of globulin is preferable.But it can be seen that degree of hydrolysis at 7%-8% from this data Oxidation resistance is most strong.

Table 2：The DH values of different enzyme hydrolysis globulin hydrolysates

	Pepsin	Trypsase	Coagulation protease trypsin	Stomach+trypsase	Stomach+pancreas+coagulation protease trypsin
						-NH₂Content (μm ol/mL)	46.87	51.67	33.26	66.58	41.45
N content (mg/mL)	9.77	9.88	5.64	11.09	6.01
						DH (%)	5.41	6.34	7.62	7.83	9.61

3.6 stomaches+pancreas+coefficient result of pancreas coagulated protein protease and analysis

It will be obtained after ultrafiltration<3kD、3kD-10kD、>The peptide fragment of 10kD different molecular weight sizes, bee is obtained after freeze-drying The enzymolysis product of the different peptide fragments of royal jelly each component.Then compound concentration is the royal jelly enzymolysis of the different peptide fragments of 0.01mg/mL Product.Its anti-oxidant degree is surveyed with DPPH free radical methods.

The royal jelly globulin intersected by three kinds of protease after digesting can phase to the elimination ability of DPPH free radicals To increase.However each ultrafiltration component of royal jelly globulin to DPPH free radical scavenging activities with royal jelly molecular weight of albumen Reduce and constantly increase.It is less than 3kD that inhibiting rate is highest in three peptide fragments, is secondly 3kD-10kD, most weak is big In 10kD's.

Table 3：Stomach-pancreas-coefficient enzymolysis product of pancreas coagulated protein protease is to the inhibiting rate of DPPH

	After enzymolysis	X ＞ 10kD	3kD ＜ X ＜ 10kD	X ＜ 3kD
					Stomach+pancreas+coagulation protease trypsin solution product (%)	24.43	20.1	24.33	28.47

4 conclusions

The content of globulin in the black bee royal jelly in 4.1 Xinjiang accounts for 1/3 or so of royal jelly dry weight, with expected results phase Symbol.

The antioxidant activity of 4.2 royal jelly protein hydrolysates is more than non-enzymolysis product, right in preceding four kinds of enzymolysis products The Scavenging activity of DPPH is best for coagulation protease trypsin, is secondly pepsin+trypsase, trypsase is most unstable Be pepsin.Intersect elimination ability of the royal jelly globulin after digesting to DPPH free radicals by three kinds of protease It can opposite increase.

4.3 survey antioxidant activity measurement result with inhibition Linoleic Acid Oxidation method is consistent substantially with above-mentioned.Preceding four kinds of sides In the enzymolysis solution product of formula enzymolysis, inhibiting rate highest, that is, antioxidant activity of stomach+trypsin digestion product is most strong, is secondly pancreas Coagulated protein enzyme enzymolysis product antioxidant activity, trypsin digestion product antioxidant activity, pepsin zymolyte it is anti-oxidant Ability is most weak.

4.4 find enzymolysis product after ultrafiltration is divided into three peptide fragments, the most strong peptide for being less than 3kD of antioxidant activity Class substance, secondly the peptide matters of 3kD-10kD, the antioxidant activity more than 10kD segments is very weak.It is risen in royal jelly globulin The substance of antioxidation is mainly the peptides less than 3kD.

4.5, which inhibit Linoleic Acid Oxidation method to survey antioxidant activity, total antioxidant activity measurement result and DPPH, removes method survey Oxidation resistance measurement result is consistent.

4.6 protein are and very stable easily with coomassie brilliant blue staining, measure the protein solution of various concentration gradient Absorbance after, calculate its degree of correlation R²=0.9997, it can be deduced that the recovery rate in globulin extraction process is very high, gained ball Globulin purity in albumen is very high.Enzymolysis degree highest of the degree of hydrolysis at 7%-8%, oxidation resistance are most strong.

Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair The restriction of embodiments of the present invention for those of ordinary skill in the art, may be used also on the basis of the above description To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is every to belong to this hair The obvious changes or variations that bright technical solution is extended out are still in the row of protection scope of the present invention.

Claims

1. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang, which is characterized in that including walking as follows Suddenly：

(1) the black bee royal jelly freeze-dried powder in Xinjiang is prepared；

(2) degreasing and the main albumen of royal jelly is prepared；

(3) globulin is prepared；

2. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 1, It is characterized in that, the black bee royal jelly freeze-dried powder preparation method in Xinjiang is as follows in step (1)：

Fresh black bee royal jelly is taken, after being weighed with assay balance, is put into centrifuge tube, then will be placed with fresh black bee royal jelly Centrifuge tube is placed in -75~-85 DEG C of refrigerators, after fresh black bee royal jelly fully charge, is freezed with vacuum freeze drier dry It is dry to get the black bee royal jelly freeze-dried powder in Xinjiang.

3. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 1, It is characterized in that, in step (2), degreasing with the following method：By the black bee royal jelly freeze-dried powder petroleum ether in Xinjiang temperature be 3 1~3h to be stirred under conditions of~5 DEG C, with heat collecting type constant temperature blender with magnetic force, carries out degreasing, filtering discards filtrate and obtains filter residue, then Filter residue petroleum ether is repeated into degreasing three times under similarity condition, last obtained filter residue is degreasing royal jelly.

4. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 3, It is characterized in that, in step (2), the method for preparing the main albumen of royal jelly is as follows：

Degreasing royal jelly is dissolved in distilled water and extracts, filter, then filter residue distilled water is repeated into extraction three under similarity condition It is secondary, filtrate is then combined with, gained filtrate is royal jelly water solubility total protein solution, and royal jelly water solubility total protein solution is used After 14K dialysis membranes are dialysed, reservation liquid is put into -75~-85 DEG C of refrigerators and is freezed, is freeze-dried with vacuum freeze drier Afterwards up to the main protein freeze-dried powder of royal jelly；Filter residue prepares raw material for globulin.

5. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 1, It is characterized in that, in step (2), using one-step method degreasing and prepares water-soluble total protein, be as follows：

Bis- (2- ethoxys) amino (trihydroxy methyl) methane and N, N- (2- ethoxys) -2- amino second are sequentially added in distilled water Sulfonic acid makes a concentration of 0.3~0.5g/L, the N of bis- (2- ethoxys) amino (trihydroxy methyl) methane, N- (2- ethoxys) -2- ammonia A concentration of 0.1~0.2g/L of base ethanesulfonic acid, stirring, the pH for adjusting solution are 7 to get royal jelly activation extraction liquid；Then to Royal jelly activation extraction liquid adds in the black bee royal jelly freeze-dried powder in Xinjiang, and 0.5~1h of stir-activating adds petroleum ether, every liter of bee Royal jelly activation extraction liquid adds in Xinjiang 4~7g of black bee royal jelly freeze-dried powder, the volume ratio of petroleum ether and royal jelly activation extraction liquid It is 1:1.5~2；0.5~1h, filtering are vibrated, filter residue prepares raw material for globulin；Filtrate stratification removes organic phase；By water After mutually being dialysed with 14K dialysis membranes, reservation liquid is put into -75~-85 DEG C of refrigerators and is freezed, is freezed with vacuum freeze drier Up to the main protein freeze-dried powder of royal jelly after drying.

6. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 1, It is characterized in that, in step (3), in temperature under conditions of 4 DEG C, by degreasing and prepares the filter residue after the main albumen of royal jelly with 8 The NaCl solution dissolving of~12 times of volumes, a concentration of 1.5~2.5wt%, with heat collecting type constant temperature blender with magnetic force stirring 1.5~ Filter residue is repeated extraction three times, merging filtrate by 2h, filtering with the NaCl solution of a concentration of 1.5~2.5wt% under similarity condition It is dialysed, reservation liquid is put into after freezing in -75~-85 DEG C of refrigerators, after being freeze-dried with vacuum freeze drier with dialysis membrane afterwards Gained dry powder is globulin.

7. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 1, It is characterized in that, in step (3), in temperature under conditions of 4 DEG C, by degreasing and prepares the filter residue after the main albumen of royal jelly with 8 ~12 times of volumes, NaCl concentration be 1.5~2.5wt%, a concentration of 0.3~0.6wt% of 4- hydroxyethyl piperazineethanesulfonic acids, pH= 7 solution dissolving stirs 0.5~1h with heat collecting type constant temperature blender with magnetic force, and filtrate with dialysis membrane is dialysed, will retained by filtering Liquid is put into freeze in -75~-85 DEG C of refrigerators after, vacuum freeze drier freeze-drying after gained dry powder be globulin.

8. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 1, It is characterized in that, in step (4), weighs after 0.2500g globulin is dissolved in 62.5mL ultra-pure waters, be divided into five parts：

First part is then placed in 37 DEG C of constant temperature cyclotron oscillations with precision acidity meter tune pH to 2.0, addition 0.0667g pepsins 4h is shaken in device or is put into 2h in globulin batch enzymolysis experimental provision；

Second part is then placed in 37 DEG C of constant temperature cyclotron oscillations with precision acidity meter tune pH to 7.5, addition 0.0333g trypsase 4h is shaken in device or is put into 2h in globulin batch enzymolysis experimental provision；

Third part is then placed in 37 DEG C of constant temperature convolutions and is shaken with precision acidity meter tune pH to 8.5, addition 0.0333g coagulation protease trypsins It swings concussion 4h in device or is put into 2h in globulin batch enzymolysis experimental provision；

4th part is first adjusted to pH to 2.0 with precision acidity meter, adds in 0.0667g pepsins, is put into 37 DEG C of constant temperature cyclotron oscillations 2h is shaken in device or is put into 1h in globulin batch enzymolysis experimental provision, is then added in again with precision acidity meter tune pH to 7.5 0.0333g trypsase is put into concussion 2h in 37 DEG C of constant temperature cyclotron oscillation devices or is put into globulin batch enzymolysis experimental provision Interior 1h；

5th part, first with precision acidity meter tune pH to 2.0, adds in 0.1000g pepsins, is put into 37 DEG C of constant temperature cyclotron oscillation devices Interior concussion 2h is put into 1h in globulin batch enzymolysis experimental provision, later with precision acidity meter tune pH to 7.5, adds 0.1000g trypsase is put into concussion 2h in 37 DEG C of constant temperature cyclotron oscillation devices or is put into globulin batch enzymolysis experimental provision Interior 1h, then pH is adjusted to 8.5 with precision acidity meter, 0.1000g coagulation protease trypsins are eventually adding, are put into 37 DEG C of constant temperature cyclotron oscillations 2h is shaken in device or is put into 1h in globulin batch enzymolysis experimental provision；

Above-mentioned five parts of solution is positioned over boiling water bath 15min in digital display type thermostat water bath after enzymolysis, inactivates enzyme；Respectively With centrifuge, centrifugal force 12000xg obtains enzymolysis product after centrifuging 10min at 4 DEG C：It is obtained after first part of centrifugation Enzymolysis product be named as pepsin enzymolysis product, second part centrifugation after obtain enzymolysis product be named as trypsin digestion production It obtains obtaining enzymolysis product after enzymolysis product is named as coagulation protease trypsin enzymolysis product, the 4th part of centrifugation after object, the centrifugation of third part It is named as after stomach+trypsin digestion product, the 5th part of centrifugation and obtains enzymolysis product and be named as stomach+pancreas+coagulation protease trypsin enzymolysis Product；

By enzymolysis product pepsin enzymolysis product obtained above, trypsin digestion product, coagulation protease trypsin enzymolysis product, Stomach+trypsin digestion product and stomach+pancreas+coagulation protease trypsin enzymolysis product with molecular cut off are 10kD and the super filter tube of 3kD Or the royal jelly enzymolysis product ultrafiltration apparatus that molecular cut off is 10kD and 3kD carries out ultra-filtration and separation and obtains the peptide more than 10kD Section, the peptide fragment of 3kD-10kD and the peptide fragment less than 3kD.

9. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 8, It is characterized in that, globulin batch enzymolysis experimental provision includes globulin storing containers (1-1), globulin digests container, Micropump (1-14), enzymolysis protease storing containers (1-2) and globulin enzymolysis product storing containers (1-17), the ball egg White enzymolysis container includes housing (1-3), level-one mixing vessel (1-4), level-one enzymolysis pipeline (1-5), level-one enzymolysis container (1- 6), two level mixing vessel (1-7), secondary enzymolysis pipeline (1-8) and secondary enzymolysis container (1-9), the housing (1-3) is interior to be equipped with Incubation cavity, the level-one mixing vessel (1-4), the level-one digest pipeline (1-5), the level-one digests container (1-6), described Two level mixing vessel (1-7), the secondary enzymolysis pipeline (1-8) and the secondary enzymolysis container (1-9) are separately positioned on described In incubation cavity；The outlet end of the globulin storing containers (1-1) and the enzymolysis protease storing containers (1-2) go out liquid The liquid feeding end fluid communication respectively with the level-one mixing vessel (1-4) is held to connect, the level-one mixing vessel (1-4) goes out liquid It holds and is connect with the liquid feeding end of level-one enzymolysis container (1-6) by level-one enzymolysis pipeline (1-5) fluid communication, described one The outlet end of grade enzymolysis container (1-6) is connect with the liquid feeding end fluid communication of the micropump (1-14), the micropump (1- 14) outlet end is connect with the liquid feeding end fluid communication of the two level mixing vessel (1-7), the two level mixing vessel (1-7) Outlet end connect with the liquid feeding end fluid communication of the secondary enzymolysis container (1-9), secondary enzymolysis container (1-9's) goes out Liquid end is connect with globulin enzymolysis product storing containers (1-17) fluid communication；The level-one enzymolysis pipeline (1-5) and institute It is the finned tube pipeline that inner wall is equipped with spoiler (1-18) to state secondary enzymolysis pipeline (1-8)；The high temperature matchmaker of the incubation cavity Jie's import is connect with the high temperature media outlet fluid communication of heating plant (1-16)；In the level-one mixing vessel (1-4) and institute It states and mixing agitating paddle (1-10) is provided in two level mixing vessel (1-7), the mixing is used with agitating paddle (1-10) with mixing The output shaft of motor (1-12) is sequentially connected；In level-one enzymolysis container (1-6) and in the secondary enzymolysis container (1-9) The output shaft transmission of motor (1-13) is used to connect with agitating paddle (1-11), the enzymolysis agitating paddle (1-11) and enzymolysis equipped with enzymolysis It connects；The outlet end and the liquid feeding end fluid of the two level mixing vessel (1-7) of the enzymolysis protease storing containers (1-2) are led Lead to and connect；Temperature sensor (1-15) there are three being set on the level-one enzymolysis pipeline (1-5)；In level-one enzymolysis pipeline (1- 5) on, first temperature sensor (1-15) is arranged on the inlet of the level-one enzymolysis pipeline (1-5), second temperature sensing Device (1-15) is arranged in the pipe of described level-one enzymolysis pipeline (1-5) terminal, and third temperature sensor (1-15) is arranged on institute State the liquid outlet of level-one enzymolysis pipeline (1-5)；Temperature sensor (1-15) there are three being set on the secondary enzymolysis pipeline (1-8)； On the secondary enzymolysis pipeline (1-8), first temperature sensor (1-15) is arranged on the secondary enzymolysis pipeline (1-8) Inlet, second temperature sensor (1-15) are arranged in the pipe of secondary enzymolysis pipeline (1-8) terminal, third temperature Sensor (1-15) is arranged on the liquid outlet of the secondary enzymolysis pipeline (1-8)；Temperature sensor (1- is equipped in the incubation cavity 15)；The level-one enzymolysis pipeline (1-5) is spiral pipeline；The secondary enzymolysis pipeline (1-8) is spiral pipeline.

10. the preparation method of the black bee royal jelly globulin enzymolysis product in antioxidant activity Xinjiang according to claim 8, It being characterized in that, enzymolysis product ultrafiltration apparatus includes the peptides separation system of the peptides separation system and 3kD-10kD more than 10kD, Peptides separation system more than 10kD includes enzymolysis product storing containers (1), the first solution feed pump (2), the first Hollow Fiber Ultrafiltration Membrane module (3), the first concentrate cut-back tank (5), the second solution feed pump (8), the first rolling hyperfiltration membrane assembly (6) and first appear Liquid storage container (4), the outlet end of the enzymolysis product storing containers (1) and the liquid feeding end fluid of first solution feed pump (2) Conducting connection, the outlet end of first solution feed pump (2) and the liquid feeding end fluid of first polysulfone hollow fiber ultrafiltration membrane system (3) Conducting connection, concentrate outlet end and the first concentrate cut-back tank of first polysulfone hollow fiber ultrafiltration membrane system (3) (5) liquid feeding end fluid communication connection, the outlet end of the first concentrate cut-back tank (5) and second solution feed pump (8) The connection of liquid feeding end fluid communication, the outlet end of second solution feed pump (8) and the first rolling hyperfiltration membrane assembly (6) into The fluid communication connection of liquid end, the peritoneal effluent outlet end of the first rolling hyperfiltration membrane assembly (6) and first solution feed pump (2) Liquid feeding end fluid communication connects；The peritoneal effluent outlet end of first polysulfone hollow fiber ultrafiltration membrane system (3) is appeared with described first The liquid feeding end fluid communication connection of liquid storage container (4)；The concentrate outlet end of the first rolling hyperfiltration membrane assembly (6) with it is big It is connected in the liquid feeding end fluid communication of 10kD peptide fragments storing containers (22)；The peritoneal effluent of the first rolling hyperfiltration membrane assembly (6) Outlet end is provided with the first flow-limiting valve (7)；The concentrate outlet end of first polysulfone hollow fiber ultrafiltration membrane system (3) and described the The liquid feeding end of one concentrate cut-back tank (5) is connected by the first mixed flow tube (10) fluid communication, first mixed flow tube (10) The diversion pipe of spoiler (11), the liquid feeding end of first mixed flow tube (10) and the first distillation water feeder are equipped with for inner wall (9) outlet end fluid communication connection；Agitating paddle is equipped in the first concentrate cut-back tank (5), the agitating paddle is with stirring The output shaft for mixing motor is sequentially connected；

The peptides separation system of 3kD-10kD includes third solution feed pump (12), the second polysulfone hollow fiber ultrafiltration membrane system (13), second Concentrate cut-back tank (15), the 4th solution feed pump (18), the second rolling hyperfiltration membrane assembly (16) and the second peritoneal effluent storage container (14), the outlet end of the first peritoneal effluent storage container (4) and the liquid feeding end fluid communication of second solution feed pump (12) connect It connects, the outlet end of second solution feed pump (12) and the liquid feeding end fluid communication of second polysulfone hollow fiber ultrafiltration membrane system (13) Connection, the concentrate outlet end of second polysulfone hollow fiber ultrafiltration membrane system (13) and the second concentrate cut-back tank (15) The connection of liquid feeding end fluid communication, the outlet end of the second concentrate cut-back tank (15) and the 4th solution feed pump (18) Liquid feeding end fluid communication connects, the outlet end of the 4th solution feed pump (18) and the second rolling hyperfiltration membrane assembly (16) into The fluid communication connection of liquid end, the peritoneal effluent outlet end of the second rolling hyperfiltration membrane assembly (16) and second solution feed pump (12) Liquid feeding end fluid communication connection；The peritoneal effluent outlet end of second polysulfone hollow fiber ultrafiltration membrane system (13) is saturating with described second Go out the liquid feeding end fluid communication connection of liquid storage container (14)；The concentrate outlet end of the second rolling hyperfiltration membrane assembly (16) It is connect with the liquid feeding end fluid communication of the peptide fragment storing containers (23) of 3kD-10kD；In the second rolling hyperfiltration membrane assembly (16) Peritoneal effluent outlet end be provided with the second flow-limiting valve (17), limited by second flow-limiting valve (17) and surpassed by second rolling The peritoneal effluent outlet end of filter membrane component (16) flows to the flow of third solution feed pump (12) liquid feeding end；Second doughnut The concentrate outlet end of hyperfiltration membrane assembly (13) and the liquid feeding end of the second concentrate cut-back tank (15) pass through the second mixed flow Manage (20) fluid communication connection, second mixed flow tube (20) for inner wall be equipped with spoiler (11) diversion pipe, described second The liquid feeding end of mixed flow tube (20) is connect with the outlet end fluid communication of after-fractionating water feeder (19).